The field of the invention is nucleoside and oligonucleotide analogues and methods for their preparation.
Nucleoside and nucleotide analogs have long been used as pharmaceutical ingredients against a variety of viruses and cancers. Currently, a number of nucleoside and nucleotide analogues are in clinical trials for several diseases.
In the cell, nucleosides and nucleotides are phosphorylated or further phosphorylated to the corresponding nucleoside triphosphates. Nucleoside triphosphates serve as inhibitors of DNA or RNA polymerases. Nucleoside triphosphates can also be incorporated into DNA or RNA, which interferes with the elongation of DNA or RNA.
Active nucleoside analogues are generally readily phosphorylated in the target cell. Corresponding nucleoside triphosphates have high affinity to catalytic sites of the polymerases and compete with the natural nucleoside triphosphates as the substrate of the polymerases.
Certain nucleoside analogues work at the nucleoside or the monophosphate level. One group of promising nucleoside analogues is the nucleosides with conformationally locked sugar moieties. It has been reported that certain conformationally locked carbocyclic nucleoside analogues demonstrated potent activity against HCMV, HSV, and EBV (Siddiqui et al. Nucleosides Nucleotides 1996, 15, 235-250; Marquez et al. J. Med. Chem. 1996, 39, 3739-3747). A conformationally locked, carbocyclic AZT 5xe2x80x2-triphosphate has been reported to be an equipotent inhibitor of HIV reverse transcriptase (Marquez et al. J. Am. Chem. Soc. 1998, 120, 2780-2789). Other nucleosides with bicyclic sugar moieties were also prepared even though no activity was found or reported (Chao et al. Tetrahedron 1997, 53, 1957-1970; Okabe et al. Tetrahedron lett. 1989, 30, 2203-2206, Hong, et al. Tetrahedron Lett. 1998, 39, 225-228).
Favorable, conformationally locked nucleosides are expected to have a positive impact on antisense oligonucleotides. Oligonucleotides, as potential antisense therapeutics, have been recognized and explored for two decades. Oligonucleotides are capable of forming double or triple helix with complementary DNA or RNA and have the ability to target the specific sequences in the viral and cancer genome. Specific binding of oligonucleotides to the DNA or RNA targets of interest would inactivate the function associated with the DNA or RNA such as replication, transcription, and translation. Therefore, viral cycles, or cancerous process can be interrupted while the normal cell cycles are not affected.
Since natural oligonucleotides are labile to the cellular and extracellular nucleases, a great deal of efforts has been made on the study of oligonucleotide modifications, especially those modifications aimed at improving nuclease resistance and binding affinity. Oligonucleotides containing certain bicyclic nucleosides have been shown to demonstrate improved nuclease stability (Leumann et al. Bioorg. Med. Chem. Letts. 1995, 5, 1231-4; Altmann et al. Tetrahedron Lett. 1994, 35, 2331-2334, 7625-7628). Recently, 2xe2x80x2-O,4xe2x80x2-C-methylene ribonucleosides, which have a locked 3xe2x80x2-endo sugar pucker, were synthesized and incorporated into oligonucleotides. Hybridization studies show that conformationally locked nucleosides can significantly enhance hybridization of modified oligonucleotides to the complementary RNA and DNA (Obika et al. Tetrahedron Lett. 1997, 38, 8735-8738; Koshkin et al. Tetrahedron 1998, 54, 3607-3630).
There is a need for new, conformationally locked nucleosides with bicyclic sugar moieties. These novel nucleosides should be useful in antiviral, anti-cancer, and other therapies. In addition, oligonucleotides composed of these novel, modified nucleosides should have desired stability to cellular nucleases and strong binding affinity to nucleic acid targets. Therefore, these oligonucleotides should be potentially useful in therapeutics and diagnostics.
Conformationally locked bicyclic-sugar nucleosides, which have a common geometrical shape, and methods for producing conformationally locked bicyclic-sugar nucleosides are described. Nucleosides are provided having bicyclic sugar moieties and oligonucleotides comprising the following formula: 
Wherein X, Y and Z are independently selected from a group of O, S, CH2, NR, Cxe2x95x90O, Cxe2x95x90CH2 or nothing, where R is selected from a group of hydrogen, alkyl, alkenyl, alkynyl, acyl; R1 is selected from a group of adenine, cytosine, guanine, hypoxanthine, uracil, thymine, heterocycles, H, OCH3, OAc, halogen, sulfonate; R2, R3 are independently selected from a group of H, OH, DMTO, TBDMSO, BnO, THPO, AcO, BzO, OP(NiPr2)O(CH2)2CN, OPO3H, PO3H, diphosphate, triphosphate; R2 and R3 together can be PhCHO2, TIPDSO2 or DTBSO2.
The novel nucleosides described herein are anticipated to be useful in antiviral, anti-cancer, and other therapies. Oligonucleotides composed of these modified nucleosides have desired physiological stability and binding affinity that enable them to be useful in therapeutics and diagnostics.
Conformationally locked nucleosides which have a 3xe2x80x2-endo sugar pucker, and methods of their preparation are provided. Processes for preparation of previously reported bicyclic nucleoside analogues cannot be applied to the novel nucleoside analogues described herein. The analogues described resulted from the successful linking between C2xe2x80x2 and C4xe2x80x2 positions of ribose in the nucleoside analogues.
As used herein, the abbreviation xe2x80x9cAcxe2x80x9d refers to acetyl; the abbreviation xe2x80x9cBnxe2x80x9d refers to benzyl; the abbreviation xe2x80x9cBzxe2x80x9d refers to benzoyl; the abbreviation xe2x80x9cDMTxe2x80x9d refers to dimethoxytrityl; the abbreviation xe2x80x9cTHPxe2x80x9d refers to tetrahydropyranyl; the abbreviation xe2x80x9cTBDMSxe2x80x9d refers to t-butyldimethylsilyl; the abbreviation xe2x80x9cTIPDSxe2x80x9d refers to tetraisopropyldisilyl; and the abbreviation xe2x80x9cDTBSxe2x80x9d refers to di(t-butyl)silyl.
1-xcex1-Methylarabinose 1, prepared according to a published procedure (Tejima et al. J. Org. Chem. 1963, 28. 2999-3003), was protected with 1,1,3,3-tetraisopropyldisiloxanyl (TIPS) at O3 and O5 to give 2, which was converted to the ketone 3 by treatment with DMSO/DCC/TFA. The subsequent Wittig reaction and removal of TIPS afforded the alkene 4 in very good yield. Compound 4 was protected with t-butyidimethylsilyl (TBS) at O5 and with benzyl (Bn) at O3 to give 5. Hydroboration of 5 was conducted with 9-BBN to give exclusively the 2-deoxy-2-hydroxymethyl derivative 6 in excellent yield. 2-deoxy-2-hydroxymethyl derivative 6 was subjected to tritylation with 4,4xe2x80x2-O-dimethoxytrityl (DMT) chloride and removal of TBS with tetrabutylammonium fluoride (TBAF) to yield 7. 
Compound 7 was oxidized to give the aldehyde 8, which was treated with formaldehyde and sodium hydroxide to yield the 4-hydroxymethyl derivative 9 in excellent yield. The mesylation of 9 and the subsequent removal of DMT afforded 10. The cyclization effected with NaH in THF and the subsequent removal of the mesyl afforded the bicyclic sugar 11. Treatment of compound 11 with acetic anhydride in the presence of DMAP yields 12, whereas treatment with acetic anhydride/acetic acid in the presence of sulfuric acid yields 13, in which the acetoxy at C1 has an inverted orientation (1-xcex2), as compared to the methoxy of 11. 
The bicyclonucleosides having the 2xe2x80x2,4xe2x80x2-bridged sugar moiety were synthesized from condensations of silylated nucleoside bases and the bicyclic sugars as shown below. The condensation of 13 with bis(trimethylsilyl)thymine yielded the product 14, the xcex1-anomer, in excellent yield. Treatment of 14 with BCl3 removed acetyl and benzyl simultaneously to yield the bicyclic xcex1-thymidine 15. 
The condensation of 13 with 6-chloro-9-trimethylsilylpurine gave a mixture of the xcex1- and xcex2-purine nucleosides, 16 and 17 (ratio of xcex1: xcex2, 1:1 to 2:3), which could be separated by chromatography. 
Treatment of 17 and 16 with ammonia in methanol, followed by hydrogenolysis, gave the adenosine analogs 18 and 19, respectively. The hydrogenolysis required a large amount of catalyst material, as well as a prolonged reaction time, because of the increased steric hindrance on the sugar moiety. Treatment of 17 and 16 with mercaptoethanol in the presence of sodium methoxide, followed by hydrogenolysis, yields inosine analogs 20 and 21, respectively. 
Condensation of 13 with the silylated N2-acetylguanine yields the xcex1-guanosine derivative 22 as the major product (30%), a small amount of the xcex2-isomer and N7-coupled products. Treatment of the xcex1-guanosine derivative with ammonia in methanol, followed by hydrogenolysis, gave the bicyclic xcex1-guanosine 23. 
As described above, the condensation reactions yielded either the xcex1-nucleoside, exclusively, or a mixture of the xcex1- and xcex2-nucleosides, without preference for the xcex2-anomers. In order to increase the ratio of xcex2-nucleosides, different condensation conditions were investigated. Temperature had little effect on the ratio of xcex1- and xcex2-anomers. However, the coupling reagent and the functional group at C1 of the sugar did have significant effects on the ratio of xcex1- and xcex2-nucleosides.
Condensation of 12 with bis- or tri(trimethylsilyl) pyrimidines in the presence of tin (IV) chloride gave the xcex2-nucleosides as major products in good yields. Thus, the reaction of 12 with silylated thymine gave the thymidine derivative 24, with xcex2:xcex1 ratio of xcx9c4:1. Condensation of 12 with the silylated uracil and N4-benzoylcytosine gave the corresponding nucleosides 25 and 26, respectively, with xcex2:xcex1 ratio of xcx9c9:1 in both reactions. Treatment of 24-26 with boron trichloride afforded the pyrimidine bicyclonucleosides 27-29, respectively. In the case of cytidine derivative, the benzoyl group of 29 was removed by treatment with ammonia to give 30. An alternative route (not shown) to prepare 30 started from 28, which was acetylated at O3xe2x80x2 and O5xe2x80x2, followed by the reaction with triazole and the subsequent treatment with ammonia. In this way, 30 was obtained in moderate yield. 
The condensation of 12 with the silylated purines, along with tin (IV) chloride as the coupling reagent, was also investigated. Unlike the reactions with pyrimi dines, the condensation of the silylated 6-chloropurine with 12 yielded not only the xcex1- and xcex2-nucleosides 16 and 17, but also an N7-coupling product (not shown). Similarly, the condensation of the silylated N2-acetylguanine with 12 yielded a mixture of three products, the N7-coupled xcex2-nucleosides 31 (42%), the desired xcex2-nucleoside 32 (10%) and the xcex1-nucleoside 22 (6%). However, when heated with the silylated N2-acetylguanine in the presence of trimethylsilyl triflate, the N7-coupled product 31 was partially converted to the N9-coupled, xcex1- and xcex2-bicyclonucleosides 22 (xcx9c22%) and 32 (xcx9c25%). The separated 32 was subjected to the same treatments as 22 to give the bicyclic xcex2-guanosine 33. 
Stereochemical assignments of the 2,6-dioxabicyclo[3,2,1]octane derivative 11 and the bicyclonucleosides formed from condensation of bicyclic sugars with silylated nucleoside bases can be assigned by NOE proton NMR. As indicated by a stick-ball model, the rigid dioxabicyclo[3,2,1]octane ring system forces the protons (H1xe2x80x2 and H2xe2x80x2) at C1xe2x80x2 and C2xe2x80x2 of the xcex1-bicyclonucleosides to become nearly parallel, whereas the H1xe2x80x2 and H2xe2x80x2 in the xcex2-bicyclonucleosides direct to the opposite sides. For example, the torsion angle of H1xe2x80x2-C1xe2x80x2-C2xe2x80x2-H2xe2x80x2 of the bicyclic xcex1-thymidine 15 after a geometry optimization is 37xc2x0 and, in consistency with this, a coupling constant of 3.9 Hz in proton NMR was observed. The torsion angle of H1xe2x80x2-C1xe2x80x2-C2xe2x80x2-H2xe2x80x2 in the bicyclic xcex2-thymidine 27 is 96xc2x0 after a geometry optimization and, as expected, no coupling between the H1xe2x80x2 and H2xe2x80x2 was observed. In fact, the proton at C1xe2x80x2 in all the xcex2-bicyclonucleosides measured is a single peak. In contrast, in all the xcex1-bicyclonucleosides measured the proton at C1xe2x80x2 is a doublet with a coupling constant of xcx9c4.0 Hz.
The stereochemical assignments of the bicyclonucleosides were further confirmed by X-ray crystal structures of the bicyclic thymidines 15 and 27. The ribose ring of the dioxabicyclo[3,2,1]octane sugar moiety in both compounds adopts a typical C3xe2x80x2-endo sugar pucker while the six-membered ring in the sugar moiety adopts the chair form. The thymine base in both compounds has the anti orientation.
The bicyclic xcex2-thymidne 27, the bicyclic xcex2-N4-benzoylcytidine 29, and the bicyclic xcex2-N4-acetylcytidine 29 were protected with DMT and then converted to the corresponding phosphoramidites, respectively. Because of the steric hindrance, a longer reaction time was required. 